Alzheimer's disease (AD) is an increasingly prevalent form of neurodegeneration that accounts for approximately 50%-60% of the overall cases of dementia among people over 65 years of age. It currently affects an estimated 15 million people worldwide and, owing to the relative increase of elderly people in the population, its prevalence is likely to increase over the next 2 to 3 decades. AD is a progressive disorder with a mean duration of around 8.5 years between onset of clinical symptoms and death. Death of pyramidal neurons and loss of neuronal synapses in brain regions associated with higher mental functions results in the typical symptoms, characterized by gross and progressive impairment of cognitive function (Francis, P. T., et al., J. Neurol. Neurosurg. Psychiatry 66 (1999) 137-147). AD is the most common form of both senile and presenile dementia in the world and is recognized clinically as relentlessly progressive dementia that presents with increasing loss of memory, intellectual function and disturbances in speech (Merritt, A Textbook of Neurology, 6th ed., Lea& Febiger, Philadelphia (1979), pp. 484-489). Neuropathologically, the major hallmarks of AD are the presence of two characteristic lesions: the amyloid senile plaque and neurofibrillary tangle (NFT). While the plaque is deposited extraneuronally, the tangle is observed intraneuronally in the post-mortem brain. One of the major components of the amyloid plaque core is the pathologically deposited small amyloid-beta-peptide (Aβ), which is cleaved by secretases from amyloid precursor protein (APP) (Selkoe, D. J., Physiol. Rev. 81 (2001) 741-766; Hardy, J. and Selkoe, D. J., Science 297 (2002) 353-356; Bush, A. I. and Tanzi, R. E., Proc. Natl. Acad. Sci. USA 99 (2002) 7317-7319). Aβ (Abeta), a self-aggregating peptide of 39-43 residues (MW ˜4 kDa), is synthesized as part of the larger APP (110-120 kDa). APP is a type I integral membrane glycoprotein with a large N-terminal extracellular domain, a single transmembrane domain and a short cytoplasmic tail. The Aβ region spans portions of the extracellular and transmembrane domains of APP. The most common hypothesis for the participation of APP in neuronal cell death in AD is the amyloid hypothesis. This hypothesis postulates that plaque amyloid depositions or partially aggregated soluble Aβ trigger a neurotoxic cascade, thereby causing neurodegeneration similar to AD pathology (Selkoe, D. J., Physiol. Rev. 81 (2001) 741-766; Hardy, J. and Selkoe, D. J., Science 297 (2002) 353-356).
Human insulin-like growth factor I (IGF-I) is a circulating hormone structurally related to insulin. IGF-I was traditionally considered the major mediator of the actions of growth hormone on peripheral tissues. IGF-I consists of 70 amino acids and is also named somatomedin C and defined by SwissProt No. P01343. Use, activity and production are mentioned in, e.g., le Bouc, Y., et al., FEBS Lett. 196 (1986) 108-112; de Pagter-Holthuizen, P., et al., FEBS Lett. 195 (1986) 179-184; Sandberg Nordqvist, A. C., et al., Brain Res. Mol. Brain. Res. 12 (1992) 275-277; Steenbergh, P. H., et al., Biochem. Biophys. Res. Commun. 175 (1991) 507-514; Tanner, J. M., et al., Acta Endocrinol. (Copenh.) 84 (1977) 681-696; Uthne, K., et al., J. Clin. Endocrinol. Metab. 39 (1974) 548-554; EP 0 123 228; EP 0 128 733; U.S. Pat. No. 5,861,373; U.S. Pat. No. 5,714,460; EP 0 597 033; WO 02/32449; WO 93/02695.
The regulation of IGF-I function is quite complex. In the circulation, only 0.2% of IGF-I exists in the free form whereas the majority is bound to IGF-binding proteins (IGFBP's), which have very high affinities to IGF's and modulate IGF-I function. The factor can be locally liberated by mechanisms releasing IGF-I such as proteolysis of IGFBPs by proteases.
IGF-I plays a paracrine role in the developing and mature brain (Werther, G. A., et al., Mol. Endocrinol. 4 (1990) 773-778). In vitro studies indicate that IGF-I is a potent non-selective trophic agent for several types of neurons in the CNS (Knusel, B., et al., J. Neurosci. 10 (1990) 558-570; Svrzic, D., and Schubert, D., Biochem. Biophys. Res. Commun. 172 (1990) 54-60), including dopaminergic neurons (Knusel, B., et al., J. Neurosci. 10 (1990) 558-570) and oligodendrocytes (McMorris, F. A., and Dubois-Dalcq, M., J. Neurosci. Res. 21 (1988) 199-209; McMorris, F. A., et al., Proc. Natl. Acad. Sci. USA 83 (1986) 822-826; Mozell, R. L., and McMorris, F. A., J. Neurosci. Res. 30 (1991) 382-390)). U.S. Pat. No. 5,093,317 mentions that the survival of cholinergic neuronal cells is enhanced by administration of IGF-I. It is further known that IGF-I stimulate peripheral nerve regeneration (Kanje, M., et al., Brain Res. 486 (1989) 396-398) and enhance ornithine decarboxylase activity U.S. Pat. No. 5,093,317). U.S. Pat. No. 5,861,373 and WO 93/02695 mention a method of treating injuries to or diseases of the central nervous system that predominantly affects glia and/or non-cholinergic neuronal cells by increasing the active concentration(s) of IGF-I and/or analogues thereof in the central nervous system of the patient. WO 02/32449 is directed to methods for reducing or preventing ischemic damage in the central nervous system of a mammal by administering to the nasal cavity of the mammal a pharmaceutical composition comprising a therapeutically effective amount of IGF-I or biologically active variant thereof. The IGF-I or variant thereof is absorbed through the nasal cavity and transported into the central nervous system of the mammal in an amount effective to reduce or prevent ischemic damage associated with an ischemic event. EP 0 874 641 claims the use of an IGF-I or an IGF-II for the manufacture of a medicament for treating or preventing neuronal damage in the central nervous system, due to AIDS-related dementia, AD, Parkinson's Disease, Pick's Disease, Huntington's Disease, hepatic encephalopathy, cortical-basal ganglionic syndromes, progressive dementia, familial dementia with spastic parapavresis, progressive supranuclear palsy, multiple sclerosis, cerebral sclerosis of Schilder or acute necrotizing hemorrhagic encephalomyelitis, wherein the medicament is in a form for parenteral administration of an effective amount of said IGF outside the blood-brain barrier or blood-spinal cord barrier.
Reduction of brain and serum levels of free IGF-I has been related to the pathogenesis of sporadic and familial forms of AD. Furthermore, IGF-I protects neurons against Aβ-induced neurotoxicity (Niikura, T., et al., J. Neurosci. 21 (2001) 1902-1910; Dore, S., et al., Proc. Natl. Acad. Sci. USA 94 (1997) 4772-4777; Dore, S., et al., Ann. NY Acad. Sci. 890 (1999) 356-364). Recently, it was shown that peripherally administered IGF-I is capable of reducing brain Aβ levels in rats and mice (Carro, E., et al., Nat. Med. 8 (2002) 1390-1397). Furthermore, the study demonstrated that in a transgenic AD mouse model prolonged IGF-I treatment significantly reduced brain amyloid plaque load. These data strongly support the idea that IGF-I is able to reduce brain Aβ levels and plaque-associated brain dementia by clearing Aβ from the brain.
Covalent modification of proteins with poly(ethylene glycol) (PEG) has proven to be a useful method to extend the circulating half-lives of proteins in the body (Hershfield, M. S., et al., N. Engl. J. Med. 316 (1987) 589-596; Meyers, F. J., et al., Clin. Pharmacol. Ther. 49 (1991) 307-313; Delgado, C., et al., Crit. Rev. Ther. Drug Carrier Syst. 9 (1992) 249-304; Katre, Advanced Drug Delivery Reviews 10 (1993) 91-114; EP-A 0 400 472; Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69; Satake-Ishikawa, R., et al., Cell Struct. Funct. 17 (1992) 157-160; Katre, N. V., et al., Proc. Natl. Acad. Sci. USA 84 (1987) 1487-1491; Tsutsumi, Y., et al., Jpn. J. Cancer Res. 85 (1994) 9-12; Inoue, H., et al., J. Lab. Clin. Med. 124 (1994) 529-536; Chamow, S. M., et al., Bioconjugate Chem. 5 (1994) 133-140).
Other advantages of PEGylation are an increase of solubility and a decrease in protein immunogenicity (Katre, N. V., J. Immunol. 144 (1990) 209-213). A method for the PEGylation of proteins is the use of poly(ethylene glycol) activated with amino-reactive reagents like N-hydroxysuccinimide (NHS). With such reagents poly(ethylene glycol) is attached to the proteins at free primary amino groups such as the N-terminal α-amino group and the E-amino groups of lysine residues. A limitation of this approach is that proteins typically contain a considerable amount of lysine residues and therefore the poly(ethylene glycol) groups are attached to the protein in a non-specific manner at all of the free E-amino groups, resulting in a heterologous product mixture of random PEGylated proteins. Therefore, NHS-PEGylated proteins can be unsuitable for commercial use because of low specific activity. Inactivation results from covalent modification of one or more lysine residues or the N-terminal amino residue required for biological activity or from covalent attachment of the poly(ethylene glycol) residues near or at the active site of the protein. For example, it was found that modification of human growth hormone using NHS-PEGylation reagents reduces the biological activity of the protein by more than 10-fold (Clark, R., et al., J. Biol. Chem. 271 (1996) 21969-21977). Human growth hormone contains 9 lysines in addition to the N-terminal amino acid. Certain of these lysines are located in regions of the protein known to be critical for receptor binding (Cunningham, B. C., et al., Science 254 (1991) 821-825). In addition, the modification of erythropoietin by the use of amino-reactive poly(ethylene glycol) reagents results also in a nearly complete loss of biological activity (Wojchowski, D. M., et al., Biochim. Biophys. Acta 910 (1987) 224-232). Covalent modification of Interferon-α2 with amino-reactive PEGylation reagents results in 40-75% loss of bioactivity (U.S. Pat. No. 5,382,657). A similar modification of G-CSF results in greater than 60% loss of activity (Tanaka, H., et al., Cancer Res. 51 (1991) 3710-3714) and of Interleukin-2 in greater than 90% loss of bioactivity (Goodson, R. J., and Katre, N. V., BioTechnology 8 (1990) 343-346).
WO 94/12219 and WO 95/32003 claim polyethylene glycol conjugates comprising PEG and IGF or a cystein mutated IGF, said PEG attached to said mutein at a free cystein in the N-terminal region of the mutein. WO 2004/60300 describes N-terminally PEGylated IGF-I.
The recognition site of the IgA Protease is described as Yaa-Pro.!.Xaa-Pro (as used herein, “.!.” refers to the cleavage site for IgA Protease). Yaa stands for Pro (or rarely for Pro in combination with Ala, Gly or Thr: Pro-Ala, Pro-Gly, or Pro-Thr. Xaa stands for Thr, Ser or Ala (Pohlner, J., et al., Bio/Technology 10 (1992) 799-804; Pohlner, J., et al., Nature 325 (1987) 458-462; and U.S. Pat. No. 5,427,927). Natural cleavage sites have been identified by Wood, S. G. and Burton J., Infect. Immun. 59 (1991) 1818-1822. Synthetic peptide substrates for the immunoglobulin A1 protease from Neisseria gonorrhoea (type 2) are the autoproteolytic sites Lys-Pro-Ala-Pro.!.Ser-Pro (SEQ ID NO: 30), Val-Ala-Pro-Pro.!.Ser-Pro (SEQ ID NO: 31), Pro-Arg-Pro-Pro.!.Ala-Pro (SEQ ID NO: 32), Pro-Arg-Pro-Pro.!.Ser-Pro (SEQ ID NO: 33), Pro-Arg-Pro-Pro.!.Thr-Pro (SEQ ID NO: 34) and the IgA1 Cleavage Sites Pro-Pro-Thr-Pro.!.Ser-Pro (SEQ ID NO: 35) and Ser-Thr-Pro-Pro.!.Thr-Pro (SEQ ID NO: 36).
WO 2006/066891 discloses conjugates consisting of an insulin-like growth factor-1 (IGF-I) variant and one or two poly(ethylene glycol) group(s), characterized in that said IGF-I variant has an amino acid alteration at up to three amino acid positions 27, 37, 65, 68 of the wild-type IGF-I amino acid sequence so that one or two of said amino acids is/are lysine and amino acid 27 is a polar amino acid but not lysine, is conjugated via the primary amino group(s) of said lysine(s) and said poly(ethylene glycol) group(s) have an overall molecular weight of from 20 to 100 kDa is disclosed. Such conjugates are useful for the treatment of neurodegenerative disorders like Alzheimer's Disease. WO 2006/074390 refers to IGF-I fusion polypeptides.